This proposal is a multidisciplinary, multiinstitutional project to improve and simplify molecular tests for mycobacteria in clinical laboratories, and to define critical molecular and genetic factors. involved in mycobacterial virulence and resistance. The studies aim to improve diagnosis and treatment of mycobacterial tuberculosis (MTB) and of mycobacteria other than tuberculosis (MOTT) to facilitate rational vaccine development, and to devise clinically useful tests that can differentiate human hosts with active disease from those with dormant infection or distant past exposure. Currently, the number of MTb infections has increased at an alarming rate due to multiple factors, including the surge in HIV, drug use, inner city poverty, and homelessness. The emergence of multiple drug-resistant MTB strains makes the need for improved control extremely urgent. Worldwide, MTB infects more than 1 billion people and is the leading cause of infectious disease death. Methods for rapid and specific diagnosis of MTB is the most pressing need in efforts to eradicate this disease. DNA detection methods have proved extremely sensitive in research laboratories, but have not yet been widely applied in clinical testing facilities primarily due to: (a) the tedium and expense of sample preparation, and (b) the presence of inhibitors in clinical specimens. To circumvent these difficulties, we propose to (1) immunoseparate and immunoconcentrate mycobacteria from clinical specimens prior to PCR and/or other molecular detection formats; (2) enhance and simplify confirmation of PCR products and other nucleic acids in a format consisting of exonuclease conversion of DNA to single strands, followed by double hybridization to capture and detection probes. These procedures for mycobacteria detection will be optimized on a bank of mycobacteria- positive clinical sputum specimens, previously collected and characterized at Grady Memorial Hospital, the public hospital that provides care to a large indigent and minority population of the Atlanta, Georgia, metropolitan area. Additional prospective specimens will be tested from Grady and from the Atlanta VA. Drug-resistant specimens will be tested for deletions/mutations in genes related to MTB drug susceptibility, including katG , a recently defined pharmacologic target of INH. Molecular methods will also be used to identify genes that are preferentially expressed when MTB is growing within macrophages and in guinea pig animal models. Such genes may encode products essential to intracellular survival and replication. The genes will be cloned and analyzed phenotypically in a well-established macrophage virulence model using E. coli, M. smegmatis, and MTB H37Ra recombinant DNA clones. Virulence factors would be expected to be highly effective targets for vaccines, and to be useful in diagnostic tests aimed at identifying active disease.